The present invention generally relates to compositions useful for selectively lysing one or more components of blood while also achieving the stabilization of other components and, more particularly, for lysing erythrocytes while affording the stabilization necessary to preserve antigenic sites on the surface of leukocytes.
The ability to differentiate and phenotype blood cells is useful for evaluating disease states and other health conditions in living beings. One popular technique for cell differentiation and lymphocyte immunophenotyping is flow cytometry. With flow cytometry, cells from an appropriately prepared blood sample, are passed one at a time through a flow cell, which is adapted for sensing or detecting impedance changes, light scatter or some other characteristic of the cell. Some flow cytometry instruments are equipped with detectors for measuring emissions from fluorescent tags that may be associated with the cells, while other detectors measure scatter intensity or pulse duration. Data about cells that pass through the flow cell can be plotted on a cytogram according to the measured property.
During the flow cytometry process, it is important that interference from the presence of erythrocytes (red blood cells) in the blood sample be avoided. Accordingly, during sample preparation, which may be done by manual, semi-automated or automated techniques), it is popular to employ a lytic reagent for lysing red blood cells and thereby isolating the leukocyte (white blood cell) populations. Leukocytes are known to include a myeloid fraction of monocytes and granulocytes (neutrophils, basophils and eosinophils) and a lymphoid fraction (namely NK, B and T cell lymphocytes). Each of the lymphocyte populations can be distinguished based upon the distinctive cell surface antigens or markers. Moreover, within each category of lymphocytes, there are sub-categories, such as xe2x80x9chelperxe2x80x9d T cells or xe2x80x9csuppressorxe2x80x9d T cells, the latter of which also includes several subsets, distinguishable by their respective surface markers. With flow cytometry of properly prepared cells, using polyclonal or monoclonal antibodies, it is possible to assay lymphocytes to analyze cells in the various subcategories.
For instance, to prepare a sample for fluorescent flow cytometry, according to one conventional practice, a volume of fresh sample blood is provided, and a suitable amount of a desired fluorochrome labeled antibody is added. The sample and antibody mixture is incubated to allow antibody/antigen bindings to take place. After incubation, a lytic reagent (some of which are regarded as potentially toxic, i.e., those containing formaldehyde) is added to lyse erythrocytes in the sample. The debris from the lysing of the erythrocytes is optionally removed, by washing, leaving a sample of leukocytes with antibodies bound to cells with complementary surface antigens. The sample is fixed and run through a fluorescent detecting flow cytometry instrument.
Among the items of potential interest to the present invention are Brown et al, xe2x80x9cFlow Cytometry: Principles and Clinical Applications in Hematologyxe2x80x9d, Clinical Chemistry 46:8(B), 1221-1229 (2000); U.S. Pat. Nos. 4,654,312; 4,902,613; 5,030,554; 5,188,935; 5,196,182; 5,250,438; 5,260,048; 5,459,073; 5,460,797; 5,731,206; 5,776,709; 5,811,099; 5,849,517, 5,939,326; 6,110,730; and commonly owned, co-pending U.S. patent application Ser. No. 09/500,248 (xe2x80x9cFixative System, Method and Composition for Biological Testingxe2x80x9d), the teachings of each of which are hereby expressly incorporated by reference for all purposes.
Accordingly, in view of the above, there is a need in the art for an improved lytic reagent that lyses red blood cells systematically and reproducibly preserves the surface antigen characteristics of leukocytes; that enables fixing of the leukocytes during the erythrocyte lysing step; that is nontoxic; and that can be used to prepare samples for analysis beyond one day.
As will be appreciated by one skilled in the art, the above needs are met individually or in combination, and other advantages are possible, by the employment of one or more novel compositions, each of which include effective amounts of a lytic agent, an agent for minimizing lysing of white blood cells (e.g., lipoprotein) and optionally a preservative. The composition of the present invention is particularly suited for use as a lytic reagent system for flow cytometry, but may also find suitable application in other analytical systems, such as hematology analyzers and microscopy.